Of the three ras genes, kras is most frequently mutated in human cancer. Ras proteins are highly homologous but differ extensively in their C-terminal hypervariable regions that direct post-translational modifications (e.g. farnesylation) and membrane targeting. We discovered a post-translational modification unique to K-Ras: protein kinase C (PKC) mediated phosphorylation. We found that phosphorylation of K-Ras at serine 181 partially neutralized the adjacent polybasic stretch of amino acids and thereby activated a farnesyl-electrostatic switch that resulted in release of K-Ras from the plasma membrane and association with intracellular membranes, including the endoplasmic reticulum (ER), Golgi apparatus and the outer mitochondrial membrane. Most intriguing, K-Ras translocation to internal membranes was associated with cell death. Bryostatin 1, a potent PKC agonist, showed anti-tumor activity that was dependent on K-Ras serine 181. In the first cycle of this grant we proposed to expand upon these discoveries with three Aims: 1) Regulation of the farnesyl-electrostatic switch, 2) Mechanisms of phospho-K-Ras mediated apoptosis and 3) The Role of C-terminal phosphorylation of K-Ras in mouse tumor models. Much progress has been made on each aim. Most exciting are our discoveries that K-Ras signals for cell death from the cytoplasmic face of the ER, that Bcl-XL is required for phospho-K-Ras mediated cell death, and that phospho-K-Ras forms a trimolecular complex with Bcl-XL and the IP3 receptor (IP3R) and regulates the calcium channel activity. We have also been successful in constructing a double knock-in mouse that harbors a conditional oncogenic K-Ras allele that lacks the phosphorylation site at amino acid 181 (LSL-K-Ras12D181A). In this competing renewal application we propose to continue our studies with three aims: Aim 1: Regulation of the IP3 Receptor (IP3R) by phospho-K-Ras. We will characterize both structurally (protein-protein interactions) and functionally (electrophysiology) the molecular interactions between phospho-K-Ras, Bcl-XL and IP3R. We will ascertain if phospho-K-Ras alters mitochondrial calcium homeostasis. We will determine if IP3R, calpain and autophagy are required for phospho-K-Ras mediated cell death. Aim 2: Analysis of K-Ras Phosphorylation at Serine 181 in vivo. We will use our newly created LSL-K-Ras12D181A mice in two Cre- driven tumor models to test the hypothesis that phosphorylation at serine 181 negatively regulates K-Ras oncogenicity and we will use the same models to show that the efficacy of bryostatin 1 depends on phosphorylation of serine 181. Aim 3: Analysis of K-Ras Phosphorylation at Serine 181 in Human Tumor Cells. We will correlate susceptibility of human tumor cells lines with K-Ras mutation status and generate isogenic lines of human tumor cells with and without a phosphorylation site at position 181. We anticipate that our mechanistic studies of the cell biology of phospho-K-Ras along with our in vivo and human tumor cell analyses will reveal unique features of this important oncogene that can be exploited in developing anti-cancer drugs.